The X-Ray Crystal Structures of Human

The X-ray Crystal Structures of Human ␣-Phosphomannomutase 1 Reveal the Structural Basis of Congenital Disorder of Glycosylation Type 1a* Received for publication, February 16, 2006, and in revised form, March 8, 2006 Published, JBC Papers in Press, March 15, 2006, DOI 10.1074/jbc.M601505200 Nicholas R. Silvaggi‡, Chunchun Zhang§, Zhibing Lu‡, Jianying Dai§, Debra Dunaway-Mariano§, and Karen N. Allen‡1 From the ‡Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118 and the §Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87103-0001

Congential disorder of glycosylation type 1a (CDG-1a) is a con- abnormalities. Numerous clinical genotypes have been identified, with genital disease characterized by severe defects in nervous system the range of phenotypes extending from mild to severe (9, 10). CDG-1a development. It is caused by mutations in ␣-phosphomannomutase is caused by mutations in the gene encoding ␣-phosphomannomutase (of which there are two isozymes, ␣-PMM1 and ␣-PPM2). Here we (␣-PMM) (11–13). The most common mutant, R141H (40% of disease report the x-ray crystal structures of human ␣-PMM1 in the open alleles of CDG-1a patients), has a high carrier frequency of 1/70 in the conformation, with and without the bound substrate, ␣-D-mannose general population. Individuals homozygous for this mutation have not 1-phosphate. ␣-PMM1, like most haloalkanoic acid dehalogenase been identified, suggesting that this genotype is lethal (12). superfamily (HADSF) members, consists of two domains, the cap Human ␣-PMM, which catalyzes the conversion of D-mannose and core, which open to bind substrate and then close to provide a 6-phosphate (Man-6-P) to ␣-D-mannose 1-phosphate (␣-Man-1-P) solvent-exclusive environment for catalysis. The substrate phos- (see Fig. 1) is required for GDP-mannose and dolichol-phosphate-man- phate group is observed at a positively charged site of the cap nose biosynthesis. Two isoforms are found; PMM2 is expressed in all domain, rather than at the core domain phosphoryl-transfer site tissues, whereas expression of PPM1 (65% sequence identity with 2؉ 19 defined by the Asp nucleophile and Mg cofactor. This suggests PMM2) is restricted to the brain and lungs (14). ␣-PMM belongs to the that substrate binds first to the cap and then is swept into the active haloalkanoic acid dehalogenase superfamily (HADSF), distinguishing it site upon cap closure. The orientation of the acid/base residue both structurally and mechanistically from the phosphomanno/phos- Asp21 suggests that ␣-phosphomannomutase (␣-PMM) uses a dif- phoglucomutase enzymes of the phosphohexomutase superfamily that ferent method of protecting the aspartylphosphate from hydrolysis are integral to glycolysis in prokaryotes and eukaryotes (15) and to algi- than the HADSF member ␤-phosphoglucomutase. It is hypothe- nate biosynthesis in Gram-negative bacteria (16). sized that the electrostatic repulsion of positive charges at the inter- Within the HADSF, ␣-PMM is distinguished from the bacterial face of the cap and core domains stabilizes ␣-PMM1 in the open ␤-phosphoglucomutase (␤-PGM) in its C(1) anomer specificity as well conformation and that the negatively charged substrate binds to the as in the position and fold of the cap domain which serves as a lid over cap, thereby facilitating its closure over the core domain. The two the conserved catalytic core domain. In HADSF members, wherein isozymes, ␣-PMM1 and ␣-PMM2, are shown to have a conserved phosphatases predominate, the conserved aspartate nucleophile of the active-site structure and to display similar kinetic properties. Anal- ysis of the known mutation sites in the context of the structures core domain mediates the transfer of the phosphoryl group. Remark- ␣ ␤ reveals the genotype-phenotype relationship underlying CDG-1a. ably, the -PMM and -PGM evolved independently to arrive at an adaptation of the phosphatase scaffold, which allows the aspartylphosphate of the core domain to discriminate between solvent and the sugar- Glycoproteins play fundamental roles in humans (1, 2) where they are phosphate substrate as the phosphoryl group acceptor. ␣ active in cell recognition and adhesion, development, and homeostasis Herein we report the x-ray crystallographic structures of -PMM1 in 2ϩ (3–7). Defects in protein glycosylation lead to a variety of disease states the Mg -bound form and in complex with the substrate of the reac- ␣ known collectively as congenital disorders of glycosylation (CDG).2 The tion, -Man-1-P. These structures are analyzed in the context of the ␣ most common CDG, congenital disorder of glycosylation type 1a coordinates for uncomplexed -PMM2 deposited in the Protein Data (CDG-1a) (8), manifests in infancy with physical and developmental Bank by the Center for Eukaryotic Structural Genomics (accession code 2AMY) to identify active-site residues with roles in binding and catalysis as well as residues involved in subunit dimerization. The structure and * This work was supported by Grant GM61099 from the National Institutes of Health ␣ ␣ (NIH) (to K. N. A. and D. D.-M.) and NIH Training Grant HL07291 (to N. R. S.). The costs of kinetic analyses of -PMM1 and -PMM2 provide for the first time publication of this article were defrayed in part by the payment of page charges. This insight into how quaternary structure, stability, and catalytic efficiency article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. are altered in clinically important mutants. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2FUC and 2FUE) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers Uni- EXPERIMENTAL PROCEDURES versity, New Brunswick, NJ (http://www.rcsb.org/). 1 To whom correspondence should be addressed: 715 Albany St., R-702, Boston, MA Materials—Except where indicated, all chemicals were obtained 02118. Tel.: 617-638-4398; Fax: 617-638-4273; E-mail: [email protected]. 2 The abbreviations used are: CDG, congenital disorders of glycosylation; CDG-1a, con- from Sigma. Primers, T4 DNA ligase, and restriction enzymes were genital disorder of glycosylation type 1a; ␣-PMM, ␣-phosphomannomutase; ␣-Man- from Invitrogen. The Geneclean spin kit and the Qiaprep spin miniprep ␣ 1-P, -D-mannose 1-phosphate; HADSF, haloalkanoic acid dehalogenase superfamily; kit were from Qiagen. Host cells were purchased from Novagen. The ␤-PGM, ␤-phosphoglucomutase, ␣-PMM1⅐Man-1-P, ␣-PMM1 in complex with ␣-D- mannose 1-phosphate; SeMet, selenomethionine; DTT, dithiothreitol. Biolmol green phosphate assay kit was purchased from Biolmol

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FIGURE 1. Scheme for the reaction catalyzed by human ␣-phosphomannomutase. The C-1 and C-6 positions on the hexose ring are labeled in the first step.

␣ ϫ Research Laboratories Inc. The -mannose 1,6-bisphosphate used in pared by mixing 200 ml of 20 M9(10gofNH4Cl, 30 g of KH2PO4,60g

the kinetic assays was prepared according to published procedure (13). of Na2HPO4, 500 ml of H2O, pH 7.5), 20 ml of amino acid mix I (the 20 Cloning, Expression, and Purification—The cDNA encoding the standard amino acids excluding Met, Tyr, Trp, and Phe, each at 4 ␣-PMM1 gene was purified from the host cells purchased from ATCC mg/ml), 40 ml of amino acid mix II (Tyr, Trp, and Phe, each at 2 mg/ml Ј (ATCC 7483527). Oligonucleotide primers (5 -CGCGGACCTG- in distilled/deionized H2O, pH 9.0), 1 ml of vitamin mixture (riboflavin, CAGCCATGGCA, and 5Ј-ACACACAGATGTGGGATCCGGTCA), niacinamide, pyridoxine monohydrochloride, and thiamine, each at 1 containing the restriction endonuclease cleavage sites NcoI and BamHI mg/ml), 4 ml of 1 M MgSO4, 4 ml of 12.5 mg/ml FeSO4,20mlof40% (underlined), were used to amplify the ␣-PMM1 open reading frame. (w/w) glucose, and 4 ml of 10 mg/ml selenomethionine in a final volume The open reading frame of ␣-PMM2 was obtained from the Mamma- of 2 liters. Chloramphenicol (16.5 mg/liter) and kanamycin (50 mg/liter) lian Genome Collection, Homo sapiens, and amplified using primers were added to the culture. The culture was grown at 30 °C with shaking Ј containing NdeI and a BamHI restriction sites (underlined) (5 - at 200 rpm until the A reached 0.8–1.1 (24 h), at which point Ј Ј 600 GAAACTGGGCATATGGCAGCGCCT-3 and 5 -TGTGGGAG- ␣-PMM1 expression was induced by adding IPTG to a final concentra- Ј ␣ ␣ GATCCTCGGCTCT-3 ). The -PMM1 and -PMM2 genes were tion of 0.4 mM. The cells were grown for an additional 10 h at 25 °C and ϩ ϩ cloned into pET-28b( ) or pET-23b( ) vectors (Stratagene), respec- harvested by centrifugation. The protein was purified using the proce- ϩ ϩ tively. The ligation products (pET-28b( )-pmm1 or pET-23b( )- dure described above, except that the DTT concentration was increased pmm2) were used to transform Escherichia coli JM109 competent cells. to 10 mM, and 1 mM EDTA was added throughout. The purified plasmids were sequenced by the Tufts University Core The purification of ␣-PMM2 was similar to that of ␣-PMM1 with Facility. a few modifications. The cell pellet from a 10-liter culture of E. coli ␣-PMM-1 was purified from E. coli RosettaTM (DE3) cells containing RosettaTM (DE3) cells (30 g) containing the pET-23b(ϩ)-pmm2 plas- the pET-28b(ϩ)-pmm1 plasmid. Transformed cells (4L) were grown at mid was suspended in 300 ml of Tris-HCl buffer (pH 8.5, 10 mM MgCl , 37 °C in Luria broth (or minimal medium containing selenomethionine) 2 5mM DTT). The cell lysate was loaded directly onto a Q-Sepharose containing 50 ␮g/ml ampicillin and induced for 4 h with 0.4 mM isopro- column (Amersham Biosciences), pre-equilibrated with Tris-HCl pyl ␤-D-thiogalactopyranoside. Cells were harvested by centrifugation buffer (pH 8.5, 10 mM MgCl ,1mM DTT). The fractions containing and the pellet (11 g) was suspended in 100 ml of ice-cold lysis buffer (50 2 ␣-PMM2 were pooled, and (NH ) SO was added to 15%. The fractions mM Hepes, pH 7.5, 5 mM DTT). Cells were lysed by sonication (6 ϫ 6 4 2 4 were then loaded onto a phenyl-Sepharose column (Amersham Bio- min at 50% power and 30% duty cycle) with a Branson sonifier 250 sciences), pre-equilibrated with Tris-HCl buffer containing 15% (VWR Scientific Inc.). The cell lysate was centrifuged at 4 °C for 60 min (NH ) SO . The protein was eluted with a 600-ml 15% to 0% (NH ) SO at 18,000 rpm (38,384 ϫ g). The protein precipitated with 40–50% 4 2 4 4 2 4 ϫ gradient. The fractions having ␣-PMM activity were pooled and con- (NH4)2SO4 was harvested by centrifugation at 15,000 rpm (26,895 g) for 15 min. Following dissolution in 25 ml of lysis buffer and dialysis centrated, then dialyzed against 50 mM Hepes, pH 7.2, 5 mM MgCl2,1 mM DTT at 4 °C. against buffer A (50 mM Hepes, pH 7.5, 0.5 mM DTT) overnight, the ␣ protein was loaded onto a 100-ml DEAE-Sepharose column (Amer- Crystallization and Data Collection—Crystals of SeMet -PMM1 sham Biosciences) pre-equilibrated with buffer A. The column was were grown by the hanging-drop vapor-diffusion method over well washed with 100 ml of buffer A, and eluted with a 0.7-liter linear gradi- solution containing 15–18% polyethylene glycol 3350, 0.15 MDL-malate, ent of 0–0.5 M NaCl in buffer A. Fractions containing ␣-PMM1 were pH 7.0, 50 mM MgCL2,and8mM 2-mercaptoethanol. Drops were ␮ combined and (NH ) SO was added to 1 M. The solution was centri- formed by mixing 2 l of protein solution (10 mM Hepes, pH 7.5, 100 4 2 4 ␮ fuged at 15,000 rpm (26,895 ϫ g) for 5 min to remove precipitated mM NaCl, 5 mM MgCl2) at 15–25 mg/ml with 2 l of well solution. protein. The supernatant was loaded onto a 25-ml butyl-Sepharose col- Crystals were transferred to paratone N and flash-cooled in a gaseous N stream at 100 K. X-ray diffraction data were collected at the National umn (Amersham Biosciences) pre-equilibrated with 1 M (NH4)2SO4 in 2 ␣ buffer A. The column was washed with 50 ml 1 M (NH4)2SO4 in buffer A Synchrotron Light Source, Beamline X12C. -PMM1 crystallized in ϭ ϭ ϭ and then eluted with a 200-ml linear gradient of 1 to 0 M (NH4)2SO4 in space group P43212 with unit cell dimensions a b 50.9 Å, c 213.0 buffer A. ␣-PMM1 was concentrated and stored at Ϫ80 °C. The protein, Å. The 0.7 ϫ 0.3 ϫ 0.3-mm crystal diffracted to 2.1 Å resolution. The shown to be Ͼ95% pure by SDS-PAGE, was obtained in a yield of 2 mg ligand-bound structure was obtained by soaking SeMet ␣-PMM1 crys- of protein/g of cell paste. tals for1hinmother liquor containing 100 mM ␣-D(ϩ)-mannose Selenomethionine-substituted ␣-PMM1 was expressed in E. coli 1-phosphate. All data sets were indexed and scaled using DENZO and BL21-CodonPlus cells. The cells were grown in minimal medium pre- SCALEPACK (17).

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TABLE 1 Data collection and refinement statistics SeMet ␣PMM1 Inflection Edge point Remote ␣-Man-1-P-bound Resolution (Å) (last shell)a 50.0–2.10 (2.18–2.10) 50.0–1.75 (1.81–1.75) Wavelength (Å) 0.9797 0.9794 0.9500 1.1000

Space group P43212 P43212 Cell dimensions (Å) a ϭ b ϭ 51.7, c ϭ 216.0 a ϭ b ϭ 51.4, c ϭ 214.7 Reflections Observed 339,621 337,677 337,985 393,048 Unique 17,978 17,984 18,017 28,735 Completeness (%)a 99.0 (99.0) 99.1 (99.0) 99.1 (99.0) 94.8 (75.5) a,b Rmerge (%) 5.6 (40.5) 6.4 (44.8) 6.5 (49.4) 5.0 (45.5) I/␴(I)a 45.3 (8.8) 44.5 (7.9) 43.4 (7.5) 42.4 (4.0) f ؅/f ؆ (e؊) Ϫ7.0/3.5 Ϫ6.0/5.0 Ϫ3.0/2.5 Figure of merit (%) 61.7 Refinement statistics Native ␣-Man-1-P-bound No. of protein/water atoms per asu 1,971/171 1,987/235 Number of reflections (work/free) 14,980/868 25,766/1,464

Rwork/Rfree (%) 19.1/20.6 20.5/24.1 Average B-factor (Å2) Protein atoms 34.1 31.2 ␣-D-Mannose 1-phosphate na 28.9 Mg(II) ions 27.9 27.0 Water 43.8 42.9 Root mean square deviations Bond lengths (Å) 0.009 0.008 Bond angles (°) 1.22 1.17 a Values in parentheses apply to the high resolution shell indicated in the resolution row. b r ϭ͚(I Ϫ͗I͘)2/͚ I 2.

Structure Determination and Model Refinement—Because structure velocity of ␣-PMM catalyzed Glc-6-P formation from ␣-Glc-1-P was determination by molecular replacement was unsuccessful, the struc- measured in the presence of 50 ␮M ␣-Glc-1,6-bisP, 0.2 mM NADP, and ture was phased by the multiple wavelength anomalous diffraction 5 units/ml Glc-6-P dehydrogenase by monitoring the increase in Ϫ1 Ϫ1 method, using three 2.1 Å SeMet data sets. The selenium substructure absorbance at 340 nm (⌬⑀ ϭ 6.2 mM cm ) resulting from the Glc- was solved using BnP (18), which found three of the four selenium sites 6-P dehydrogenase catalyzed reduction of NADP. The initial velocity of in the protein. The sites were confirmed by comparison with the anom- ␣-PMM-catalyzed Man-6-P formation from ␣-Man-1-P was measured alous difference Patterson map. The N-terminal methionine, along with in the presence of 50 ␮M ␣-Man-1,6-bisP (13), 5 units/ml glucose-6- the next 10 residues, was disordered. An initial model was built using phosphate dehydrogenase, 5 units/ml phosphomannose isomerase, 5 RESOLVE (19) and improved by manual rebuilding in the graphics suite units/ml phosphoglucose isomerase by monitoring the increase in Ϫ1 Ϫ1 COOT (20). This partial model was refined in the REFMAC5 (21) mod- absorbance at 340 nm (⌬⑀ ϭ 6.2 mM cm ) resulting from the Glc- ule of the CCP4 suite (22) using the Hendrickson-Lattman coefficients 6-P dehydrogenase-catalyzed reduction of NADP. The kcat and Km val- from BnP. Electron density maps calculated from the combined phases ues were determined from the initial velocity data using the equation ϭ ϩ ␣ ␣ were of very high quality and permitted rapid rebuilding of the model. V0 Vm[A]/(Km [A]), where [A]isthe -Glc-1-P or -Man-1-P

Iterative cycles of restrained refinement in REFMAC5 and rebuilding in concentration, V0 is the initial velocity, Vm is the maximum velocity, and

COOT ultimately yielded a model containing 244 of 262 residues, 171 Km is the Michaelis constant. The kcat was calculated from the ratio of 2ϩ ␣ water molecules, and 2 Mg ions. The quality of the model at this point Vmax and the -PMM concentration. was assessed by MOLPROBITY (23), and 235 residues (96.3%) were in the favored regions, 9 residues were in the additionally allowed regions, RESULTS AND DISCUSSION and no residues were in the disallowed regions. Overall Structure—Recombinant human ␣-PMM1 was purified by The ligand-bound structure was solved by Fourier difference meth- conventional chromatographic techniques and crystallized from a solu- ␤ ods using phases from the 2.1 Å SeMet native model. After rigid body tion of polyethylene glycol 3350, MgCl2, -mercaptoethanol, and DL- refinement in REFMAC5 the electron density for Man-1-P was clearly malic acid at pH 7.0 for x-ray structure determination. Both the native ␣ defined. The Man-1-P was not modeled until the model reached an and SeMet -PMM1 crystallized in space group P43212 (unit cell ϭ ϭ ϭ Rwork of 22.3% and an Rfree of 27.4%. After several more rounds of model dimensions a b 50.9 Å, c 213.0 Å) with one molecule in the building and refinement, the final ␣-PMM1⅐Man-1-P model was com- asymmetric unit. Molecular replacement using the structure of prised of residues 13–258 of ␣-PMM1, 235 water molecules, two Mg2ϩ ␣-PMM2 (65% identical, Protein Data Bank accession code 2AMY, ions, and one molecule of Man-1-P. Data collection and refinement unpublished submission by the Center for Eukaryotic Structural statistics for both models are reported in Table 1. Genomics) as the search model did yield a viable solution; however, the Steady-state Kinetics—All 1-ml kinetic assays were carried out at resulting model did not refine well, and several surface loops appeared ϩ 25 °C in 50 mM K Hepes, pH 6.5, containing 10 mM MgCl2. The initial to be disordered. To obtain experimental phases, the structure was

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FIGURE 2. A, structure of human ␣-phosphomannomutase complexed with ␣-D-mannose 1-phosphate. The cap domain is magenta and the core domain cyan. Man-1-P is shown as ball-and-stick (orange) and the two Mg2ϩ ions as metallic spheres. The image was rendered using MOL- SCRIPT (38) and POVRAY. B, schematic representation of the arrangement of secondary structure elements in ␣-PMM1 core (cyan) and cap (magenta).

determined using multiple wavelength anomalous diffraction phasing primary sequence between motifs 2 and 3 (see Ref. 24 for review), place of the selenomethionine-substituted protein. The final model contained this enzyme in subclass IIb of the HADSF. The secondary structure 245 of 262 residues. The first 11 residues at the N terminus and the last elements are arranged to form a four-stranded antiparallel ␤-sheet 6 residues at the C terminus were disordered. The refined model of the (␤6-␤7 and ␤8-␤9) topped by three ␣-helices (␣4–␣6). Two flexible Mg2ϩ-bound form of ␣-PMM1 was used to phase the structure of “hinge” regions comprising residues 91–94 and 195–197 connect the ␣-PMM1 in complex with ␣-D-mannose 1-phosphate (␣-PMM1⅐Man- two domains. The hinge regions were identified using visual inspection 1-P) obtained by soaking the Mg2ϩ-bound crystals with ␣-Man-1-P. of the structure together with the program FlexOracle (25). In both ␣-PMM1, like other HADSF phosphotransferases, is comprised of structures reported herein, the cap domain is in an open conformation, two domains, a core domain and a smaller cap domain (Fig. 2). The with the active site exposed to solvent. overall dimensions of the monomer are ϳ63 ϫ 31 ϫ 42 Å. The core The 29.7-kDa monomer forms a homodimer in solution as demon- domain (residues 1–90 and 198–262) consists of a six-stranded parallel strated by size-filtration chromatography. This dimer is observed in the ␤-sheet (␤1–␤5 and ␤10-␤11) flanked by five ␣-helices (␣1-␣2 and crystal structure of ␣-PMM1, with the interface consisting exclusively ␣7–␣9) (Fig. 2B) with typical ␤-␣-␤ connections. The cap domain (res- of interactions between the cap domains (Fig. 3A). Two major interac- idues 95–194) is inserted between ␤5 and ␣7 of the core domain. The tions dominate the dimer interface: a helix-helix interaction between ␣␤␤(␣)␣␤␤ topology of the cap domain, as well as its position in the the first ␣-helices (␣4) and an antiparallel ␤-sheet interaction between

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FIGURE 3. A, ribbon representation of the ␣-PMM1 dimer. The molecule has been rotated 90° on the horizontal axis relative to Fig. 2. The cap and core domains of monomer A are magenta and cyan, respectively, while the cap and core of monomer B are red and blue, respectively. The image was rendered using MOLSCRIPT (38) and POVRAY. B, schematic representation of the interactions between monomers at the dimer interface. For clarity, the distances are shown for only the right half of the dimer interface. Green circles denote residues involved in hydrogen bonds, blue circles denote residues involved in salt bridges, and yellow circles denote residues forming the hydrophobic core of the interface. Pro122, which imparts a kinked turn necessary for positioning Arg125, is shown as a white circle.

the first ␤-strands (␤6) of the two cap domains. The interaction be- residues Arg150, Arg143, and Arg132 in ␣-PMM1 and Arg141, Arg134, and tween the strands creates an extended eight-stranded ␤-sheet across the Arg123 in ␣-PMM2. One might expect that there would be complemen- two monomers, holding the two core domains away from one another. tary negatively charged residues on the surface of the core domain; Residues Leu105, Ile109, Leu113, Phe128, Ile129, and Phe131 form a hydro- however, the corresponding surface of the core retains an overall posi- phobic core at the dimer interface. Several hydrogen bonds and a salt tive charge contributed by conserved residues Arg28 and Lys58 in bridge anchor the interface at each end (Fig. 3B). The same oligomer- ␣-PMM1 and Arg21 and Lys51 in ␣-PMM2. Thus, rather than acting as ization interface can be observed in the structure of human ␣-PMM2, a “clasp” these two surfaces may act as an electrostatic wedge retaining demonstrating that this is a conserved feature of human ␣-PMMs. One the correct interdomain orientation. Indeed, the importance of these of the more common deleterious mutations of ␣-PMM2, F119L (Phe128 residues to protein function is highlighted by the fact that the mutation in ␣-PMM1), is located in the center of the dimer interface (Fig. 3B), R141H (Arg150 in ␣-PMM1) has been identified as one of the major highlighting the potential importance of the quaternary structure for deleterious polymorphic mutations of ␣-PMM2. function and/or stability of the protein. Enzyme Active Site—The active site of ␣-PMM1 is located at the Based on previous studies of catalytic cycling in other HADSF interface of the cap and core domains (Fig. 4). By analogy to other enzymes, it is known that the cap domain must dissociate from the core HADSF phosphatases, the key active-site residues are the catalytic domain to allow substrate to bind, then associate to seal the active site nucleophile Asp19, the putative acid/base residue Asp21 (contributed by from aqueous solvent (26, 27). A portion of the cap domain that would motif 1), Ser54, which helps position the phosphoryl group (contributed contact the core domain in ␣-PMM carries a large number of positively by motif 2), the salt bridge residue Lys198 (contributed by motif 3), and charged residues (Figs. 4 and 5). These include the three conserved the metal-binding residue Asp218 (contributed by motif 4). In both the

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FIGURE 4. A, a stereo view of the ␣-D-mannose 1-phosphate moiety highlighting the well defined electron density for the ligand. The Man-1-P (orange carbon atoms) is shown as ball-and-stick ͉ ͉ Ϫ ͉ ͉ with the Fo Fc omit electron density contoured at 3.0 ␴ (green wire frame). The image was rendered using MOLSCRIPT (38) and POVRAY. B, the enzyme active site with ␣-Man-1-P (ball-and-stick with gray carbons) bound. The core domain residues are shown with cyan carbon atoms, and cap domain residues have magenta carbons. Residues located in the primary substrate specificity loop are shown with green carbons, and those of the secondary substrate specificity loop have orange carbons. The Mg2ϩ ion is shown as a metallic sphere. The image was rendered using MOLSCRIPT (38) and POVRAY. C, schematic representation of the interactions between ␣-PMM1 and Man-1-P (black). The residues of the cap and core domains and the primary and secondary substrate specificity loops are colored as described for B. The distances of the interactions are given in Angstroms.

native and ␣-PMM1⅐Man-1-P structures, the acid/base residue Asp21, general acid/base residue is pinned to the linker in the cap-open con- stationed two residues upstream from the nucleophile (thus denoted formation and to the active site in the cap-closed conformation. The the Asp-plus-2 residue), is held in position for proton transfer by a bound substrate and not a water molecule functions to stabilize the hydrogen bond to residue Gln62 contributed by ␣2, as has been noted cap-closed conformation for catalytic turnover. previously for the corresponding residue in HADSF type IIA (28), type Substrate Binding—The structure of ␣-PMM1 with ␣-D-mannose IIB (26), and type III (29) phosphatases. Because Asp21 is positioned to 1-phosphate bound identifies the active-site residues contributing to activate a water molecule as well as the C(6)OH of ␣-Man-1-P for attack substrate specificity. Since this structure was obtained by soaking pre- at the aspartylphosphate, it is clear that a different mechanism for formed crystals of ␣-PMM1 with Man-1-P, the domain motions ensuring that phosphomutase activity predominates over phosphatase required to close the active site could not occur, because the enzyme activity is operative in ␣-PMM than the substrate induced-fit mecha- conformation was held fast by the crystal lattice. The Man-1-P-bound nism, which is operative in ␤-PGM. In the case of ␤-PGM, the Asp structure was surprising because it was anticipated from observations of

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FIGURE 5. The enzyme active site with Man-1-P (orange ball-and-stick) bound. A dot surface shows the overall charge of the cap and core domain surfaces at the Man-1-P-binding site. The image was rendered using CCP4MG (39).

phosphate (and phosphate analog) binding to other HADSF members phosphoenzyme (Fig. 1). For a HADSF enzyme to act as a phospho- that the electropositive environment created by the active-site Ser54, transferase, as opposed to a phosphatase, the active site of the Lys198, and Mg2ϩ would constitute a more competent phosphate-bind- phosphorylated enzyme must be capable of discriminating between the ing site than that of the cap domain (26, 27, 30). Thus, ␣-PMM1 is substrate and water as the phosphoryl group acceptor. In both subfam- unique in that the phosphorylated substrate is seen bound primarily by ily I and subfamily II HADSF enzymes, the cap domain closes on the interactions with the cap domain (Figs. 2A and 4). The electron density active site, sealing it from solvent (31, 32). In unliganded ␤-phosphoglu- ␴ ͉ ͉ Ϫ ͉ ͉ 10 corresponding to the ligand was observed at 3.0 in a Fo Fc electron comutase, the Asp acid/base is involved in hydrogen bond interac- density map (Fig. 4A). The ␣-PMM1⅐Man-1-P structure can be said to tions with the main chain amides of two residues in the flexible hinge represent a collision complex, the first encounter of enzyme and sub- region. In this orientation, the Asp10 acid/base is pointing away from the strate, and not the Michaelis complex immediately preceding the first catalytic center, which prevents general base catalysis of phosphoryl chemical step. Indeed, if the intermediate ␣-mannose 1,6-(bis)phos- transfer to water. Binding of substrate shifts the equilibrium between phate is overlaid with the Man-1-P substrate in the ␣-PMM1⅐Man-1-P the open and closed forms of the enzyme, stabilizing the closed form. complex, the C(6)-phosphate group is 10 Å from the Asp19 nucleophile Upon cap closure, the ␤-phosphoglucomutase Asp10 acid/base rotates (that is, there is no phosphate group in the phosphoryl-transfer site). into the active site to form a hydrogen bond with the C(6)OH of the The implication of this observation is that substrate initially binds to the ␤-glucose 1-phosphate. In this way, catalysis is linked to cap closure. cap and not the core domain portion of the active site. Once the sub- The unliganded and the MIP-bound structures of ␣-PMM1 suggest a strate is bound, there is presumably a change in the conformation of the unique mechanism of discrimination between substrate and solvent. hinge region that pushes the substrate into the active site as the cap The Asp21 acid/base of ␣-PMM1 is engaged in a hydrogen bond with binds to the core domain. Gln62 (2.9 Å) located on helix ␣2 of the core domain. In the cap-open The surfaces of both domains at the Man-1-P-binding site carry an conformation, Asp21 is in position to function as an acid/base catalyst in overall positive charge (Fig. 5). The residues constituting the Man-1-P- the transfer of the phosphoryl group to and from the Asp19 nucleophile. binding site are positioned on two loops of the cap domain, termed the Thus, solvent discrimination is not accomplished by coupling the posi- primary (residues 140–162) and secondary (residues 181–190) sub- tioning of the Asp21 acid/base catalyst with substrate induced cap clo- strate specificity loops. The corresponding loops have been described sure. Rather, it must rely on transition-state stabilization by the unique for BT4131, a homologous subclass IIb HADSF enzyme (26). ␣-PMM1 active-site environment created in the cap-closed conformation. How, residues Arg143 and Arg150, together with Ser188 and the main chain then, is substrate binding tied to cap closure? The ␣-PMM structures amide of Met186, form the distal phosphate-binding site (Fig. 4, B and C). suggest that the positively charged surfaces at the interface of the cap Other residues involved in Man-1-P binding are Asp190 and Arg132, and core domains (Fig. 5) observed in the unliganded enzyme may act as which hold the C-2 hydroxyl, and Arg28, the sole core domain contact, an electrostatic wedge. Binding of the negatively charged substrate which interacts with the C-4 hydroxyl. Although Arg132 is not part of would mitigate the electrostatic repulsion of the cap and core domains, either substrate specificity loop, it does directly contact the substrate. It favoring cap closure. is located on strand ␤6, which forms a major part of the dimer interface The Structural Effects of Clinically Observed Mutations—The corre- (see above) in both ␣-PMM1 and ␣-PMM2. The importance of this lation between mutation and ␣-PMM activity has been the object of residue to substrate binding and its proximity to the dimer interface only one study reported in 1999 by Pirard et al. (33). In this study, seven may explain, at least in part, the loss of activity observed when dimer- PPM2 mutants (D65Y, F119L, V129M, R141H, R162W, D188G, and ization is disrupted by genetic mutation. V231M) were purified and their in vitro activity and thermal stability A Proposal for the Mechanism of Selective Phosphoryl Transfer— briefly surveyed. The R141H and D188G mutants proved to be the most ␣-PMM, like ␤-phosphoglucomutase, catalyzes the interconversion catalytically impaired (0.4 and 2% of wild-type activity), correlating with of the hexose 1-phosphate and hexose 6-phosphate via a hexose- the severity of the heterozygous R141H/D188G phenotype, while 1,6-(bis)phosphate intermediate generated by reaction with the V231M proved to be the least stable at 40 °C. Although not biochemi-

14924 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 21•MAY 26, 2006 Structural Basis of CDG1a

FIGURE 6. Sequence alignment of human ␣-PMM1 and ␣-PMM2. Black bars denote the four motifs of the HADSF. Identical residues are boxed in red, and conservative changes are boxed in yellow. cally characterized to date, the R123G, F183S, and P113L mutations are nonetheless clinically significant (34, 35). Because of its limited tissue distribution, mutations to ␣-PMM1 have not been linked to pathology. However, the PMM1 and PMM2 isozymes are quite similar (65% identical; Fig. 6), and the variable residues that are scattered throughout the structure are found mainly at the protein surface. The overall structures of ␣-PMM1 and ␣-PMM2 are very similar with root mean square deviations of 0.65 Å (for the cap) and 1.0 Å (for the core) when the two domains are overlaid separately. The dimer interface, active-site motifs (Fig. 6), and the hinge regions are conserved. The numerous ␣-PMM2 mutation sites known to be associated with the CDG Type 1a phenotype are not localized to one particular region of the structure but are instead distributed throughout (Fig. 7). Based on their locations in the structure, the clinically important mutations can be classified on the basis of whether substrate binding and catalysis, dimerization, or protein stability are targeted. The most severe mutations, R141H (Arg150 in PMM1) and D188G (Asp197) belong in the first category, as does R123G (Arg132). As seen in the ␣-PMM1⅐Man-1-P structure, Arg150 (Arg141 in ␣-PMM2) is a crucial part of the distal phosphate-binding site, and loss of the positive charge at this location would impair substrate binding. This conclusion is in good agreement with the finding reported in Pirard et al. (14, 36) that the Km for Man-1-P ϳ increased 10-fold in the R141H mutant. The effect of the D188G ␣ 197 FIGURE 7. Ribbon representation of the -PMM1 structure showing the positions (Asp ) mutation is equally dramatic. This residue is adjacent to the (C␣ atoms) of the clinically relevant mutations (red spheres). motif 3 lysine, which is required for proper orientation of the nucleo- philic aspartate. The R123G (Arg132) mutation is also likely to effect substrate binding, because, as seen in the Man-1-P-bound structure, The last category, mutations effecting folding and/or stability of the 138 51 Arg132 of ␣-PMM1 makes direct interactions with the C-2 hydroxyl of polypeptide, includes V129M (Ile in PMM1), V44A (Val ), D65Y Man-1-P. Of the mutations that appear to disrupt the dimer interface (Asp74), and V231M (Val240). These mutations occur in the interior of (F119L (Phe128) and P113L (Pro122)), the F119L mutation would be the cap or core domains (Fig. 7) and are therefore expected to be espe- expected to have the greatest ipmact, given that this residue is a major cially detrimental to native structure. Finally, one mutation, F183S component of the hydrophobic core that comprises the dimer interface (Phe192) appears to be unique in that it may alter the interdomain (Fig. 3B). The P113L mutation might alter the turn that positions Arg125 motion required for catalysis. Phe183 is positioned at the start of the turn (Arg116 in ␣-PMM2) for formation of a salt bridge at the dimer interface. forming the second linker region, and it is possible that changes to this

MAY 26, 2006•VOLUME 281•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14925 Structural Basis of CDG1a

TABLE 2 Steady-state kinetic constants The steady-state kinetic constants measured for ␣-PMM1 and ␣-PMM2 catalyzed conversion of ␣-Glc-1-P to Glc-6-P (50 ␮M ␣-Glc-1,6-bisP cofactor) or ␣-Man-1-P to ␮ ␣ ϩ Man-6-P (50 M -Man-1,6-bisP cofactor) in solutions of 50 mM K Hepes, pH 6.5, 25 °C, containing 10 mM MgCl2. The listed error was computed for the fit of the data set to equation 1. The value reported within the parentheses was reported earlier (14, 36) for reaction solutions containing 10 ␮M mannose-1,6-bisphosphate, 25 mM Hepes, pH 7.1, 30 °C, and 5 mM MgCl2.

Substrate Enzyme kcat Km kcat/Km Ϫ1 Ϫ1 Ϫ1 s ␮MMs ␣-Man-1-P PMM1 4.39 Ϯ 0.05(2.4) 54 Ϯ 2(3.2) 8.1 ϫ 104 (8 ϫ 105) ␣-Man-1-P PMM2 3.9 Ϯ 0.1 (2.4) 16 Ϯ 2 (18) 2.5 ϫ 105 (1.5 ϫ 105) ␣-Glc-1-P PMM1 2.85 Ϯ 0.06 (2.4) 7.5 Ϯ 0.8 (5) 3.8 ϫ 105 (5 ϫ 105) ␣-Glc-1-P PMM2 1.72 Ϯ 0.02 (0.1) 13.5 Ϯ 0.4 (12) 1.3 ϫ 105 (1 ϫ 104) residue interfere with the conformational changes expected to accom- 9. Neumann, L. M., von Moers, A., Kunze, J., Blankenstein, O., and Marquardt, T. (2003) pany substrate binding. Eur. J. Pediatr 162, 710–713 ␣ ␣ 10. Noelle, V., Knuepfer, M., Pulzer, F., Schuster, V., Siekmeyer, W., Matthijs, G., and Kinetic Analysis—The structures of -PMM1 and -PMM2 are Vogtmann, C. (2005) Eur. J. Pediatr 164, 223–226 nearly identical, and the results of the kinetic assays reinforce this obser- 11. Matthijs, G., Schollen, E., Pardon, E., Veiga-Da-Cunha, M., Jaeken, J., Cassiman, J. J., vation (Table 2). In addition, the kinetic analysis establishes the integrity and Van Schaftingen, E. (1997) Nat. Genet. 16, 88–92 of the recombinant PMM enzymes. 12. Matthijs, G., Schollen, E., Van Schaftingen, E., Cassiman, J. J., and Jaeken, J. (1998) Both ␣-PMM1 and ␣-PMM2 are efficient catalysts with high speci- Am. J. Hum. Genet. 62, 542–550 13. Van Schaftingen, E., and Jaeken, J. (1995) FEBS Lett. 377, 318–320 ficity for glucose 1-phosphate and mannose 1-phosphate, as indicated 14. Pirard, M., Achouri, Y., Collet, J. F., Schollen, E., Matthijs, G., and Van Schaftingen, E. by the magnitude of kcat/Km. In two instances the observed kinetic (1999) Biochem. J. 339, 201–207 parameters differ significantly from those of previous studies (14, 36). 15. Dai, J. B., Liu, Y., Ray, W. J., Jr., and Konno, M. (1992) J. Biol. Chem. 267, 6322–6337 ␣ ␣ ␮ 16. Naught, L. E., and Tipton, P. A. (2001) Arch. Biochem. Biophys. 396, 111–118 First, the Km of -PMM1 for -Man-1-P of 54 M is larger than the 17. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326 published value of 3.2 ␮M. Nevertheless, this larger value is reasonable 18. Weeks, C. M., Blessing, R. H., Miller, R., Mungee, R., Potter, S. A., Rappleye, J., Smith, given that the average serum mannose concentration in humans is 55 G. D., Xu, H., and Furey, W. (2002) Z. Kristallogr. 217, 686–693 ␮M (range 35–70 ␮M) (37). Also, Pirard et al. (14) reported that 19. Terwilliger, T. C. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 1863–1871 ␣-PMM2 is 10-fold more reactive with ␣-Man-1-P than with ␣-Glc- 20. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 1-P, while our results indicate that there is no significant difference in 2126–2132 21. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D Biol. reactivity, consistent with the conservation of active site residues. Crystallogr. 53, 240–255 Conclusion—The structural and kinetic analyses reported here in are 22. Collaborative Computational Project, No. 4 (1994) Acta Crystallogr. Sect. D Biol. important for several reasons. First, the key structural and functional Crystallogr. 50, 760–763 sites in the two ␣-PMM isozymes are conserved. Thus, we anticipate 23. Davis, I. W., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2004) Nucleic Acids Res. 32, W615–W619 that mutations will effect both enzymes in similar manner. The clinical 24. Allen, K. N., and Dunaway-Mariano, D. (2004) Trends Biochem. Sci. 29, 495–503 manifestation, however, will depend on the isozyme tissue distribution. 25. Echols, N., Milburn, D., and Gerstein, M. (2003) Nucleic Acids Res. 31, 478–482 Second, location of the positions of the clinical mutations in the ␣-PMM 26. Lu, Z., Dunaway-Mariano, D., and Allen, K. N. (2005) Biochemistry 44, 8684–8696 structures provide a link between phenotype and genotype, which can 27. Lahiri, S. D., Zhang, G., Dunaway-Mariano, D., and Allen, K. N. (2002) Biochemistry be explored in detail through future structure/function analysis of the 41, 8351–8359 28. Tremblay, L. W., Dunaway-Mariano, D., and Allen, K. N. (2006) Biochemistry 45, mutant enzymes. Finally, the divergence of mutase function in HASDF 1183–1193 subclass II, which we contend is mechanistically distinct from that 29. Peisach, E., Selengut, J., Dunaway-Mariano, D., and Allen, K. N. (2004) Biochemistry observed in the HASDF subclass I, provides a birds eye view of the 43, 12770–12779 diversity of nature at the molecular level. 30. Morais, M. C., Zhang, W., Baker, A. S., Zhang, G., Dunaway-Mariano, D., and Allen, K. N. (2000) Biochemistry 39, 10385–10396 31. Wang, W., Cho, H. S., Kim, R., Jancarik, J., Yokota, H., Nguyen, H. H., Grigoriev, I. V., Acknowledgments—We gratefully acknowledge the use of the data collection Wemmer, D. E., and Kim, S. H. (2002) J. Mol. Biol. 319, 421–431 facilities at Beamline X12C of the National Synchrotron Light Source at 32. Zhang, G., Dai, J., Wang, L., Dunaway-Mariano, D., Tremblay, L. W., and Allen, K. N. Brookhaven National Laboratories. 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